Diff of 6.4.1 Antibody staining in fins

The added line is THIS COLOR.
The deleted line is THIS COLOR.
Go to 6.4.1 Antibody staining in fins.
[[FrontPage]]

*6.4.1 Antibody staining in fins

Yuki Nakatani~
Sae Sakaguchi~
Tokyo Institute of Technology

**6.4.1.1 Reagents

-4% paraformaldehyde
-PBST: PBS + 0.1% Triton X-100
-methanol
-2xMAB: 0.2M maleic acid, 0.3M NaCl, adjust pH to 7.5
-1xMAB
-MABD: 1xMAB, 10mg/ml BSA, 1% DMSO, 0.1% Triton X-100. Make fresh before use.
-MABDN: 2% FCS (or goat/sheep serum) in MABD

**6.4.1.2 Method

+Fins are fixed with 4% paraformaldehyde at 4&ordm;C, O/N.
+Wash with PBST.
+Dehydrate gradually through a methanol series (25-50-75-100% methanol in PBST).
+Store in 100% methanol at -20&ordm;C (several minutes to hours¡Ä).
+Rehydrate gradually through PBST.
+1xMAB wash at RT for 5 min.
+0.1% TritonX-100/1xMAB at 4&ordm;C for 15 min, three times.
+MABD at 4 oC for 30 min, two times.
+MABDN at 4 oC for 30 min.
+primary antibody (ex. alpha-acetylated tubulin: 1/1000) in MABDN.
+4&ordm;C, O/N or RT for 2 hours with rotate.
+MABD at 4&ordm;C for 5 min, three times.
+MABDN at 4&ordm;C for 30 min, four times.
+secondary antibody (ex. coupled to FITC or Alexa 488) in MABDN.
+4&ordm;C, O/N or RT for 2 hours in the dark with rotate.
+MABD at 4&ordm;C for 5 min in the dark, three times.
+MAB at 4&ordm;C for 30 min in the dark, four times.
+Mount and observation.